Caveolin-1 Modulates Cardiac Gap Junction Homeostasis and Arrhythmogenecity by Regulating cSrc Tyrosine Kinase Kai-Chien Yang, Cody A. Rutledge, Mao Mao, Farnaz R. Bakhshi, An Xie, Hong Liu, Marcelo G. Bonini, Hemal H. Patel, Richard D. Minshall and Samuel C. Dudley, Jr Circ Arrhythm Electrophysiol. 2014;7:701-710; originally published online July 13, 2014; doi: 10.1161/CIRCEP.113.001394 Circulation: Arrhythmia and Electrophysiology is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 1941-3149. Online ISSN: 1941-3084
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Data Supplement (unedited) at: http://circep.ahajournals.org/content/suppl/2014/07/13/CIRCEP.113.001394.DC1.html
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Original Article Caveolin-1 Modulates Cardiac Gap Junction Homeostasis and Arrhythmogenecity by Regulating cSrc Tyrosine Kinase Kai-Chien Yang, MD, PhD; Cody A. Rutledge, PhD; Mao Mao, MS; Farnaz R. Bakhshi, PhD; An Xie, PhD; Hong Liu, MD, PhD; Marcelo G. Bonini, PhD; Hemal H. Patel, PhD; Richard D. Minshall, PhD; Samuel C. Dudley Jr, MD, PhD Background—Genome-wide association studies have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Methods and Results—Using adult Cav1-/- mice, we revealed a marked reduction in the left ventricular conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the activation of cSrc tyrosine kinase, resulting in the downregulation of connexin 43 and subsequent electric abnormalities. Pharmacological inhibition of cSrc mitigates connexin 43 downregulation, slowed conduction, and arrhythmia inducibility in Cav1-/- animals. Using a transgenic mouse model with cardiac-specific overexpression of angiotensin-converting enzyme (ACE8/8), we demonstrated that, on enhanced cardiac renin–angiotensin system activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation at Cys156, leading to cSrc activation, connexin 43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. Renin–angiotensin system–induced Cav1 S-nitrosation was associated with increased Cav1–endothelial nitric oxide synthase binding in response to increased mitochondrial reactive oxidative species generation. Conclusions—The present studies reveal the critical role of Cav1 in modulating cSrc activation, gap junction remodeling, and ventricular arrhythmias. These data provide a mechanistic explanation for the observed genetic link between Cav1 and cardiac arrhythmias in humans and suggest that targeted regulation of Cav1 may reduce arrhythmic risk in cardiac diseases associated with renin–angiotensin system activation. (Circ Arrhythm Electrophysiol. 2014;7:701-710.) Key Words: arrhythmias, cardiac ◼ caveolin 1 ◼ connexin 43 ◼ renin–angiotensin system
H
uman genetic studies have revealed an important link between caveolins and cardiac arrhythmias.1–3 Among the genes (CAV1, CAV2, and CAV3) encoding the 3 distinct caveolin isoforms named caveolins 1 to 3, mutations in CAV3 have been shown to lead to congenital long-QT1 and sudden infant death4 syndromes, whereas human genome-wide association studies have observed significant association of CAV1 variants with PR intervals2 and increased susceptibility to cardiac arrhythmias.2,3 It is clear now that caveolin-3 (Cav3) interacts with and regulates cardiac sodium channel Nav1.5,1 and that mutations in CAV3 can lead to a 3- to 5-fold increase in late sodium currents, resulting in delayed repolarization, prolonged QT intervals, and arrhythmogenic phenotype.1,4 In contrast, albeit caveolin 1 (Cav1) is known to express in cardiomyocytes5,6 and has been implicated in regulating ion channels in in vitro studies,7,8 there is no established mechanism explaining the genetic link between CAV1 and cardiac
arrhythmias. We sought to determine the mechanistic link between Cav1 and cardiac arrhythmias.
Clinical Perspective on p 710
Methods Animals were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols involving animals were approved by the Animal Studies Committee at the University of Illinois at Chicago, Lifespan, or the Veterans Administration San Diego Healthcare System. In vivo electrophysiological studies, including ECG recordings, programmed stimulation, and ventricular conduction velocity, were performed on Cav1-/- and angiotensin-converting enzyme (ACE8/8) mice (all in C57/Bl6 background) that were derived and maintained as described previously.9–11 Left ventricular (LV) tissue and cardiomyocytes isolated from Cav1-/-, Cav3-/-, and ACE8/8 mice were used for Western blotting, immunoprecipitation, S-nitrosation assay, NO measurement, and transcript analyses.
Received November 11, 2013; accepted June 10, 2014. From the Lifespan Cardiovascular Research Center, Department of Medicine, Warren Alpert School of Medicine, Brown University, Providence Veterans Administration Medical Center, RI (K.-C.Y., C.A.R., A.X., H.L., S.C.D.); Department of Medicine (K.-C.Y., C.A.R.), Department of Pharmacology (M.M., M.G.B., R.D.M.), and Department of Anesthesiology (F.R.B., R.D.M.), University of Illinois at Chicago; and Department of Anesthesiology, VA San Diego Healthcare Systems, University of California (H.H.P.). The Data Supplement is available at http://circep.ahajournals.org/lookup/suppl/doi:10.1161/CIRCEP.113.001394/-/DC1. Correspondence to Samuel C. Dudley, MD, PhD, Lifespan Cardiovascular Institute, The Warren Alpert Medical School of Brown University, 593 Eddy St, APC 730, Providence, RI. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Arrhythm Electrophysiol is available at http://circep.ahajournals.org
DOI: 10.1161/CIRCEP.113.001394
Downloaded from http://circep.ahajournals.org/ 701 at Tulane University on September 1, 2014
702 Circ Arrhythm Electrophysiol August 2014 All measurements were presented in dot plots with mean±SEM. The inducibility of ventricular tachycardia was presented as percentage of all tested animals in the same group. The statistical significance of differences between experimental groups was evaluated by the exact version of the Mann–Whitney U test or Fisher exact test, followed by Holm test to correct for multiple comparisons; P values
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circep.ahajournals.org/content/7/4/701
Data Supplement (unedited) at: http://circep.ahajournals.org/content/suppl/2014/07/13/CIRCEP.113.001394.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation: Arrhythmia and Electrophysiology can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation: Arrhythmia and Electrophysiology is online at: http://circep.ahajournals.org//subscriptions/
Downloaded from http://circep.ahajournals.org/ at Tulane University on September 1, 2014
Original Article Caveolin-1 Modulates Cardiac Gap Junction Homeostasis and Arrhythmogenecity by Regulating cSrc Tyrosine Kinase Kai-Chien Yang, MD, PhD; Cody A. Rutledge, PhD; Mao Mao, MS; Farnaz R. Bakhshi, PhD; An Xie, PhD; Hong Liu, MD, PhD; Marcelo G. Bonini, PhD; Hemal H. Patel, PhD; Richard D. Minshall, PhD; Samuel C. Dudley Jr, MD, PhD Background—Genome-wide association studies have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Methods and Results—Using adult Cav1-/- mice, we revealed a marked reduction in the left ventricular conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the activation of cSrc tyrosine kinase, resulting in the downregulation of connexin 43 and subsequent electric abnormalities. Pharmacological inhibition of cSrc mitigates connexin 43 downregulation, slowed conduction, and arrhythmia inducibility in Cav1-/- animals. Using a transgenic mouse model with cardiac-specific overexpression of angiotensin-converting enzyme (ACE8/8), we demonstrated that, on enhanced cardiac renin–angiotensin system activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation at Cys156, leading to cSrc activation, connexin 43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. Renin–angiotensin system–induced Cav1 S-nitrosation was associated with increased Cav1–endothelial nitric oxide synthase binding in response to increased mitochondrial reactive oxidative species generation. Conclusions—The present studies reveal the critical role of Cav1 in modulating cSrc activation, gap junction remodeling, and ventricular arrhythmias. These data provide a mechanistic explanation for the observed genetic link between Cav1 and cardiac arrhythmias in humans and suggest that targeted regulation of Cav1 may reduce arrhythmic risk in cardiac diseases associated with renin–angiotensin system activation. (Circ Arrhythm Electrophysiol. 2014;7:701-710.) Key Words: arrhythmias, cardiac ◼ caveolin 1 ◼ connexin 43 ◼ renin–angiotensin system
H
uman genetic studies have revealed an important link between caveolins and cardiac arrhythmias.1–3 Among the genes (CAV1, CAV2, and CAV3) encoding the 3 distinct caveolin isoforms named caveolins 1 to 3, mutations in CAV3 have been shown to lead to congenital long-QT1 and sudden infant death4 syndromes, whereas human genome-wide association studies have observed significant association of CAV1 variants with PR intervals2 and increased susceptibility to cardiac arrhythmias.2,3 It is clear now that caveolin-3 (Cav3) interacts with and regulates cardiac sodium channel Nav1.5,1 and that mutations in CAV3 can lead to a 3- to 5-fold increase in late sodium currents, resulting in delayed repolarization, prolonged QT intervals, and arrhythmogenic phenotype.1,4 In contrast, albeit caveolin 1 (Cav1) is known to express in cardiomyocytes5,6 and has been implicated in regulating ion channels in in vitro studies,7,8 there is no established mechanism explaining the genetic link between CAV1 and cardiac
arrhythmias. We sought to determine the mechanistic link between Cav1 and cardiac arrhythmias.
Clinical Perspective on p 710
Methods Animals were handled in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols involving animals were approved by the Animal Studies Committee at the University of Illinois at Chicago, Lifespan, or the Veterans Administration San Diego Healthcare System. In vivo electrophysiological studies, including ECG recordings, programmed stimulation, and ventricular conduction velocity, were performed on Cav1-/- and angiotensin-converting enzyme (ACE8/8) mice (all in C57/Bl6 background) that were derived and maintained as described previously.9–11 Left ventricular (LV) tissue and cardiomyocytes isolated from Cav1-/-, Cav3-/-, and ACE8/8 mice were used for Western blotting, immunoprecipitation, S-nitrosation assay, NO measurement, and transcript analyses.
Received November 11, 2013; accepted June 10, 2014. From the Lifespan Cardiovascular Research Center, Department of Medicine, Warren Alpert School of Medicine, Brown University, Providence Veterans Administration Medical Center, RI (K.-C.Y., C.A.R., A.X., H.L., S.C.D.); Department of Medicine (K.-C.Y., C.A.R.), Department of Pharmacology (M.M., M.G.B., R.D.M.), and Department of Anesthesiology (F.R.B., R.D.M.), University of Illinois at Chicago; and Department of Anesthesiology, VA San Diego Healthcare Systems, University of California (H.H.P.). The Data Supplement is available at http://circep.ahajournals.org/lookup/suppl/doi:10.1161/CIRCEP.113.001394/-/DC1. Correspondence to Samuel C. Dudley, MD, PhD, Lifespan Cardiovascular Institute, The Warren Alpert Medical School of Brown University, 593 Eddy St, APC 730, Providence, RI. E-mail [email protected] © 2014 American Heart Association, Inc. Circ Arrhythm Electrophysiol is available at http://circep.ahajournals.org
DOI: 10.1161/CIRCEP.113.001394
Downloaded from http://circep.ahajournals.org/ 701 at Tulane University on September 1, 2014
702 Circ Arrhythm Electrophysiol August 2014 All measurements were presented in dot plots with mean±SEM. The inducibility of ventricular tachycardia was presented as percentage of all tested animals in the same group. The statistical significance of differences between experimental groups was evaluated by the exact version of the Mann–Whitney U test or Fisher exact test, followed by Holm test to correct for multiple comparisons; P values
